UNIVERSITI PUTRA MALAYSIA

INTAN SHAMEHA BINTI ABDUL RAZAK

FPV 2009 19

MORPHOLOGY AND MUCOSAL IMMUNITY OF THE OVIDUCT AND UTERUS DURING FOLLICULAR AND LUTEAL PHASES IN EWES

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MORPHOLOGY AND MUCOSAL IMMUNITY OF THE OVIDUCT AND UTERUS DURING FOLLICULAR AND LUTEAL PHASES IN EWES

By

INTAN SHAMEHA BINTI ABDUL RAZAK

Thesis submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Doctor of

Philosophy

October 2009

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I wish to dedicate this thesis to my inspired late Father,

Abdul Razak bin Abdul Mutalib

Who always wanted me to, and now your wishes come true.

And to my precious children,

Aidiel Ikmal, Ariff Iskandar, Annuralisa Izmira &

Azzalea Irdina

May this masterpiece be your inspiration for your future

endeavors…

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Doctor of Philosophy

MORPHOLOGY AND MUCOSAL IMMUNITY OF THE OVIDUCT AND UTERUS DURING FOLLICULAR AND LUTEAL PHASES IN EWES

By

INTAN SHAMEHA BINTI ABDUL RAZAK

October 2009

Chairman: Md Zuki Abu Bakar @ Zakaria, PhD

Faculty: Veterinary Medicine

Reproductive diseases result in major production losses in the sheep

industry. Although extensive studies on mucosal surfaces of the female

reproductive tract had been conducted, very little information particularly

the mechanism is known. Furthermore, defining the mucosal immunity is

complicated by the critical interface between the endocrine and immune

systems. Thus, an approach to gain sight into the pathogenesis of the

reproductive diseases by investigating the local uterine cellular immune

response under the precise hormonal influences throughout the estrous

cycle was undertaken.

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Fourteen ewes were synchronized into estrus and the plasma samples were

collected every alternate day for hormonal profiles using radioimmunoassay

(RIA) techniques. The ewes were divided into two groups: follicular and

luteal phases groups (n=7).All the ewes were observed for three consecutive

cycles before they were slaughtered at the peak of the follicular and luteal

phases. The ewes started to exhibit signs of estrus between 24 to 36 hours

following the intravaginal sponges’ removal. The average estradiol level

during the peak of the follicular phase was 5.44 pg/ml for the first cycle, 4.85

pg/ml for the second and 4.25pg/ml for the third cycle. The ewes started the

luteal phase which peaked at day 9 with the average progesterone value of

4.21ng/ml and 4.65 ng/ml respectively. The estrus period occurred for 30.6

± 0.65 hours, luteal phase period ranged between 12 to 14 days, making the

complete cycle of 15 days. Average vaginal mucous resistances in ewes were

recorded for three consecutive cycles using Draminski® estrous detector. The

daily plasma concentrations of estradiol and progesterone were significantly

correlated (p<0.01) with the estrus detector reading at (r = -0.924) and (r =

0.705), respectively.

At slaughter, samples from the anterior, middle and posterior horns and the

oviducts were taken and processed accordingly for light and electron

microscopy. Under light microscopy, the number of lymphocytes in

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different parts of the uterus was significantly (p<0.05) higher during the

follicular phase compared to the luteal phase. However, in the follicular

phase group, the number of lymphocytes was not significantly different

between the middle and anterior horn, while in the luteal phase group, the

number of lymphocytes was not significantly different between the posterior

and middle horn. Similarly, the quantity of plasma cell revealed that the

number was significantly (p<0.05) higher in the follicular phase compared to

the luteal phase for the different parts of the reproductive tract. However, in

the luteal phase group, the number of plasma cells was not significantly

different between the posterior and middle horn and between the anterior

horn and oviduct.

Ampullae were taken and processed accordingly for scanning (SEM) and

transmission electron microscopy (TEM). During the follicular phase, the

population of secretory cells was less than the luteal phase, while the

number of ciliated cells was higher than the luteal phase. The secretory cells

were rounded, turgid with intact microvilli in the follicular phase, but in the

luteal phase the surfaces were broken and some secretions were oozing out.

The TEM examination revealed that during the follicular phase, the

secretory cells had blunt processes at the apex with intact microvilli, but

during the luteal phase, the cytoplasmic protrusion on the secretory cells

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exhibited an increase in volume. Numerous secretory granules with

different sizes and electron density were found in both phases. During the

follicular phase, the secretory cells were at the preparatory stage while they

were actively secreting during luteal phase. The present study revealed

marked cyclic changes and differences of the secretory cells during these

two phases of the estrous cycle. Results of current study also suggested that

the secretory granules were released by exocytosis and the apocrine would

be the mode of secretion at estrus while the merocrine is the mode of

secretion at luteal phase.

In vitro experiments were conducted on the adhesion and colonization of

E.coli to the uterus explants of both the follicular and luteal phases ewes.

Scanning electron microscopy showed there were significant (p<0.05)

differences between the groups at all different post-inoculation times except

for the negative control group and between 180 and 360 minutes post-

inoculation for both groups. Hence, the adhesion of E. coli to uterus during

follicular phase was significantly (p<0.05) lower than the luteal phase. It

seems that the intensity of adhesions increases with time.

This present study contributes to the new knowledge on the endocrinology

of Malin crossbred ewes where complete estradiol and progesterone

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hormonal profiles were obtained for the first time. In addition, the vaginal

mucous resistance data recorded using the Draminski® estrous detector

might be useful as the guideline in determining the exact timing of the estrus

phase during the estrous cycle.

Present results demonstrated that the mucosal immunity is lower during

peak luteal phase than follicular phase in the ewes since the number of

lymphocytes and plasma cells were higher during peak follicular phase. The

morphological evaluations confirmed that during follicular phase, the uterus

and the oviduct are at the preparatory stage for the fertilization process

while during luteal phase, the cellular immune response is modified to

minimize rejection in the case if the ewe conceived. The in vitro challenged of

the uterine explants confirmed the severity of the bacterial colonization

during luteal phase than the follicular phase.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Doktor Falsafah

MORFOLOGI DAN KEIMUNAN MUKOSA OVIDUKTUS DAN UTERUS SEMASA FASA FOLIKEL DAN LUTEUM PADA BIRI-BIRI

Oleh

INTAN SHAMEHA BINTI ABDUL RAZAK

Oktober 2009

Pengerusi: Md Zuki Abu Bakar @ Zakaria, PhD

Fakulti: Perubatan Veterinar

Penyakit berkaitan reproduksi telah mengakibatkan kerugian besar kepada

industri ternakan. Walaupun kajian terhadap permukaan mukosa trakus

reproduktif betina telah dijalankan secara meluas, masih sedikit maklumat

berkaitan mekanismenya diperolehi. Tambahan lagi, penakrifan keimunan

mukosa semakin rumit oleh hubungkait kritikal antara sistem endokrin dan

imun. Oleh itu, pendekatan terhadap patogenesis penyakit reproduksi ini

melalui pengkajian tindakbalas keimunan sel pada rahim di bawah

pengaruh hormon yang tepat di sepanjang kitaran estrus telah dijalankan.

Empat belas bebiri kacukan Malin telah diselaraskan masa estrusnya supaya

berlaku secara serentak, dan sampel plasma telah diambil selang sehari

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untuk mendapatkan profil hormon menggunakan teknik radioimmunoassai

(RIA). Bebiri ini dibahagikan kepada dua kumpulan: kumpulan fasa folikel

dan luteum (n=7). Kesemua bebiri ini diperhatikan untuk tiga kitaran estrus

berturut-turut sebelum mereka disembelih samada pada waktu puncak fasa

folikel atau luteum. Bebiri ini mula menunjukkan ciri-ciri biang di antara 24

hingga 36 jam selepas span intravagina dicabut. Purata paras estradiol

semasa waktu puncak fasa folikel ialah 5.44 pg/ml untuk kitaran pertama,

4.85 pg/ml untuk kitaran kedua dan 4.25 pg/ml bagi kitaran ketiga. Bebiri

ini mula berada pada fasa luteum yang memuncak pada hari ke-9 dengan

purata paras progesteron 4.21 ng/ml, 4.65ng/ml dan 4.2ng/ml untuk

kitaran pertama, kedua dan ketiga. Tempoh masa estrus berlaku selama 30.6

± 0.65 jam, dan jangkamasa fasa luteum ialah antara 12 hingga 14 hari,

menjadikan satu kitaran estrus lengkap ialah selama 15 hari. Purata bacaan

rintangan mukus pada vagina telah direkodkan bagi tiga kitaran berturut-

turut menggunakan pengesan estrus Draminski®. Kandungan estradiol dan

progesteron dalam plasma harian di dapati berkorelasi secara signifikan (r=-

0.924) dan (r=0.705) pada (P=0.01) dengan bacaan pengesan estrus

Draminski®.

Selepas disembelih, sampel dari bahagian anterior, tengah dan posterior

tanduk rahim serta oviduktus diambil dan diproses sewajarnya (mengikut

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prosedur) untuk mikroskop cahaya dan elektron. Di bawah mikroskop

cahaya, bilangan limfosit pada bahagian uterus berbeza semasa fasa folikel

adalah lebih tinggi secara signifikan (p<0.05) berbanding semasa fasa

luteum. Walaubagaimanapun, di dalam kumpulan fasa folikel, bilangan

limfosit didapati tidak berbeza secara signifikan (p>0.05) di antara tanduk

tengah dan anterior, dan di dalam kumpulan luteal, bilangan limfosit

didapati tidak berbeza secara signifikan di antara tanduk posterior dan

tengah. Begitu juga dengan kuantiti sel plasma, di mana didapati

bilangannya adalah tinggi secara signifikan (p<0.05) semasa fasa folikel

berbanding dengan fasa luteum pada bahagian-bahagian yang berbeza di

dalam saluran pembiakan. Walaubagaimanapun, di dalam kumpulan fasa

luteum, bilangan sel plasma tidak berbeza dengan signifikan di antara

tanduk posterior dan tanduk tengah serta antara tanduk anterior dan

oviduktus.

Ampula telah diambil dan diproses sewajarnya untuk mikroskop elektron

imbasan dan transmisi. Semasa fasa folikel, populasi sel rembesan adalah

berkurangan berbanding dengan semasa fasa luteum, sementara bilangan

sel silia didapati lebih banyak semasa fasa luteum. Semasa fasa folikel, sel

rembesan didapati berbentuk bulat, membengkak dan mempunyai

mikrovili pada permukaannya, tetapi pada fasa luteum, permukaan sel ini

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telah pecah dan terdapat rembesan yang mula keluar. Pemeriksaan

mikroskop elektron transmisi (TEM) mendapati semasa fasa folikel, sel

rembesan mempunyai lanjutan berbentuk tumpul dan bermikrovili pada

bahagian apeksnya, manakala, semasa fasa luteum, pertambahan unjuran

sitoplasma sel rembesan terjadi. Banyak granul rembesan yang berbilang

saiz dan densiti elektron pada kedua-dua fasa. Semasa fasa folikel, sel

rembesan di dalam keadaan bersedia dan menjadi aktif semasa fasa luteum.

Kajian ini mendapati terdapat perubahan dan perbezaan berkitar yang

ketara dan pada sel rembesan semasa kedua fasa kitaran estrus. Hasil

penemuan kajian ini juga mencadangkan granul rembesan dibebaskan

melalui proses eksositosis; dan modnya ialah secara apokrin semasa tempoh

biang manakala merokrin adalah mod rembesan semasa fasa luteum.

Eksperimen in vitro pelekatan dan pengkolonian E.coli terhadap eksplan

mukosa uterus fasa folikel dan luteum telah dijalankan. Pemeriksaan

mikroskop elektron imbasan menunjukkan terdapat perbezaan yang

signifikan (p<0.05) di antara semua kumpulan dan tempoh pascaeraman

kecuali pada kawalan negatif, dan antara 180 dan 360 minit pascaeraman

bagi kedua kumpulan. Pelekatan E. coli juga didapati lebih rendah secara

signifikan (p<0.05) semasa fasa folikel berbanding fasa luteum. Ini

menunjukkan keamatan perlekatan bertambah dengan masa.

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Hasil penemuan ini telah menyumbang kepada maklumat baru dalam

bidang endokrinologi bebiri baka kacukan Malin di mana profil hormon

estradiol dan progesteron yang lengkap telah dapat direkodkan buat

pertama kalinya. Maklumat ini dilengkapkan dengan data bacaan rintangan

mukus vagina yang direkod menggunakan alat pengesan estrus Draminski®

di mana ianya dapat dimanfaatkan sebagai panduan semasa menentukan

fasa estrus semasa kitaran estrus.

Hasil kajian ini juga telah membuktikan tahap keimunan mukosa semasa

waktu puncak fasa luteum adalah lebih rendah berbanding fasa folikel

memandangkan bilangan limfosit dan sel plasma yang lebih tinggi semasa

waktu puncak fasa folikel. Penilaian morfologi telah dapat mengesahkan

semasa fasa folikel, rahim dan oviduktus berada dalam keadaan bersedia

untuk proses persenyawaan tetapi semasa fasa luteum, tindakbalas

keimunan sel diubahsuai bagi meminimumkan penolakan sekiranya

kehamilan berlaku. Cabaran in vitro telah membuktikan pengkolonian

bakteria yang lebih teruk semasa fasa luteum berbanding fasa folikel.

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ACKNOWLEDGEMENTS

In the Name of Allah, the Most Benevolent and Most Merciful……

Alhamdulillah, I would like to express my utmost thanks and gratitude to

Almighty Allah S.W.T. who has given me the capability and strength to

complete this thesis and my ‘selawat’ and ‘salam’ to His righteous, Prophet

Muhammad (peace be upon him).

It is a pleasure to express my gratitude wholeheartedly to my supervisor,

Assoc. Prof. Dr. Md Zuki Abu Bakar, who has devoted much of his time for

invaluable guidance, advice, supervision and support throughout the course

of this study. Sincere thanks are also due to my co-supervisors, Assoc. Prof.

Dr Abd. Wahid Haron, Assoc. Prof. Dr. Noordin Mohamed Mustapha and

Prof. Dr. Tengku Azmi Tengku Ibrahim who have provided advices and

helpful discussion that have enlightened and improved this study.

I wish to express my sincere gratitude to the former Dean of Faculty of

Veterinary Medicine, UPM, Prof. Dr Mohd Zamri Saad who was

instrumental in providing me the opportunity to pursue the candidature,

and Universiti Putra Malaysia and Ministry of Higher Education for

granting me the study leave and scholarships during my study.

I am deeply indebted to Puan Maizatulakmal Moktar, Mr. Raziman

Sulaiman and Mr. Ariff Ahmad who were most helpful and had contributed

their efforts in making this study a success. Special thanks to Dr. Goh Yong

Meng for his help in statistical analysis.

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I have also been very fortunate in receiving assistance from a number of my

colleagues and friends particularly, Mr. Yap Keng Chee, Mr. Saipuzaman

Ali, Mr. Mohd Kamarudin Awang Isa, Mr Hajara’ih Salamat, Dr. Anjas

Asmara Samsudin, Mr. Abd. Rashid Abd. Rahman, Mrs. Sapiah Jalal, Miss

Kunnasunthary Ramachandran and other staffs of Histopathology, Virology

and Bacteriology laboratories, FVM, UPM.

I also benefitted from kind outstanding works of Prof. Dr. Fauziah Othman,

Mr. Ho Oi Kuan, Miss Azilah Abd Jalil, Mr Rafiuz Zaman Haroun, Mrs

Noraini Mohd Ain, Mrs Faridah Akmal Ismail, Mrs Noor Hayati Jamaludin,

and the staff members of the Electron Microscope Unit, Institute of

Biosciences, UPM.

My greatest appreciation to my beloved husband Abu Hassan, my

wonderful children, Aidiel Ikmal, Ariff Iskandar, Annuralisa Izmira and

Azzalea Irdina whom I owe the most for their patience, understanding and

support through this long and demanding project. To my mother, Puan

Ramlah Abu Kassim and mother-in-law, Puan Fadzilah Ibrahim, words fail

me to express my appreciation of your sacrifices, patience, understanding

and supports.

Last but not least, continuous moral support and encouragement from my

brothers, Zulkefli and Amir Izwan, and friends Noraniza, Mazlina, Awang

Rozaini, Hasliza, Nurul, Po Po, Lat lat, Hafandi, Mokrish, and many others

that I could not mention here are deeply appreciated.

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APPROVAL SHEET

I certify that an Examination Committee has met on 14th October 2009 to conduct the final examination of Intan Shameha Abdul Razak on her Doctor of Philosophy thesis entitled “Morphology and Mucosal Immunity of the Oviduct and Uterus during Follicular and Luteal Phases in Ewes” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the student be awarded the Doctor of Philosophy.

Members of the Examination Committee were as follows:

Mohamed Ariff bin Omar, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman) Mohamed Ali bin Rajion, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Internal Examiner) Abdul Azhar Kassim, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Arief Boerdiono, PhD Professor Faculty of Veterinary Medicine Bogor Agricultural University Indonesia (External Examiner) __________________________ BUJANG KIM HUAT, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia

Date:

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APPROVAL SHEET

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee were as follows:

Md Zuki Abu Bakar@Zakaria, PhD Associate Professor, Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman) Abdul Wahid Haron, PhD Associate Professor, Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Noordin Mohamed Mustapha, PhD Associate Professor, Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Tengku Azmi Tengku Ibrahim, PhD Professor, Faculty of Veterinary Medicine Universiti Putra Malaysia (Member)

__________________________________ HASANAH MOHD GHAZALI, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia

Date: 11 February 2010

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DECLARATION I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution. _______________________________ INTAN SHAMEHA ABDUL RAZAK Date: 25 November 2009

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TABLE OF CONTENTS

Page

DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF PLATES LIST OF ABBREVIATIONS

ii

vii xii xiv xvi xxii xxiii xxiv xxix

CHAPTER

1

2

INTRODUCTION

LITERATURE REVIEW Reproductive Cycles and Influences

Reproductive cycle Endocrine Regulation of Estrous Cycle

Follicular Dynamics Transrectal ultrasonography Folliculogenesis Follicular growth The early stage of follicular development Antral follicular waves in ewe Endocrinology of follicular growth and development Steroidogenesis Follicular growth and development during

follicular and luteal phase Ovulation in the ewe Corpus luteum formation and development Immunology of Reproduction Innate Immune System Acquired Immune System Regulation of Cellular Immune Function in the Reproductive Tract Steroid Hormone Regulation

1

6 6 7 9 9 13 13 14 15 18

18

20 21 24 26 27 28 29

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Estrogen-influence Progesterone-Influence Components of the Reproductive Tract Immune System Phagocytosis Macrophages PMN Cells Lymphocytes T-Lymphocytes B- Lymphocytes Plasma Cells Anatomic Considerations Other Components Ciliated Cells Secretory Cells

33 36 38

38 39 39 40 42 43 43 44 47 48 48

3 GENERAL MATERIALS AND METHODS Animals Estrous Synchronization Determination of estradiol and progesterone hormone profiles Sample Collection and Processing for Light Microscopic Examination Sample Collection and Processing for Scanning Electron Microscope (SEM) Sample Collection and Processing for Transmission Electron Microscope (TEM) Statistical Analysis

50

50 51 51

56

57

57

58

4 DETERMINATION OF PEAK FOLLICULAR AND LUTEAL PHASES IN EWES BY DETERMINING PROGESTERONE AND ESTRADIOL LEVELS BY USING RIA TECHNIQUE Introduction Materials and Methods

Animals Blood Samples Collection Radioimmunoassay Technique (RIA) Detection of Estrus Statistical Analysis

Results Plasma concentrations of estradiol and

59

59 62 62 62 62 63 66 66

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progesterone Mean estrous cycle length Mean estrus period Signs of estrus Confirmation of estrus Vaginal mucous resistance Discussion Conclusion

66 69 69 71 73 76 81 83

5 QUANTIFICATION OF THE LYMPHOCYTES AND PLASMA CELL POPULATION IN THE OVIDUCT AND UTERINE MUCOSAE DURING FOLLICULAR AND LUTEAL PHASES Introduction Materials and Methods Animals Experimental Procedures Sample Collection and Processing for Light microscopic Examination Haematoxylin and Eosin Staining

Morphometry evaluation Statistical Analysis

Results Histological examination of the distribution of lymphocytes

Distributions of lymphocytes in the uterus and oviduct

Histological examination of the distribution of plasma cells

Distributions of plasma cells in the uterus and Oviduct Discussion Conclusion

86

86 87 87 87 87

88 88 89 89 89

95

97

99

101 105

6 HISTOLOGICAL AND ULTRASTRUCTURAL EVALUATIONS OF THE UTERINE AND OVIDUCTAL MUCOSAE DURING FOLLICULAR AND LUTEAL PHASES Introduction Materials and Methods Animals Experimental Procedures

106

106 108 108 108

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Sample Collection and Processing for Light Microscopy (LM) Sample Collection and Processing for Scanning Electron Microscope (SEM) Sample Collection and Processing for Transmission Electron Microscope (TEM Results Histological Evaluation of the Uterus during Follicular and Luteal Phases Endometrium Endometrium lining Subepithelial region Glands Caruncles

Histological Evaluation of the Oviduct during Follicular and Luteal Phases

Ultrastructure evaluation of Oviduct by Scanning Electron Microscopy (SEM)

Ultrastructure Evaluation of Oviduct by Transmission Electron Microscopy (TEM) Follicular phase

Luteal phase Discussion Conclusion

108

108

108

108

110 110 113 117 120 123

129

135

135 140 148 155

7 IN-VITRO SUSCEPTIBILITY OF THE UTERUS TO

Escherichia coli INFECTION DURING FOLLICULAR AND LUTEAL PHASES Introduction Materials and Methods

Animals Sample Collection and Processing

Inoculum Preparation Experimental Procedures Sample Processing Scoring System Statistical Analysis

Results Adhesion to the Follicular and Luteal Phase Uterus Explants Discussion

156

156 158 158 158 158 159 160 160 161 162 162

170

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Conclusion

174

8 GENERAL DISCUSSION

176

9 SUMMARY, GENERAL CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE RESEARCH

193

BIBLIOGRAPHY APPENDICES BIODATA OF STUDENT LIST OF PUBLICATIONS

197 221 239 241